Component analysis device, drug component analysis device, component analysis method, and drug component analysis method

ABSTRACT

Provided is a component analysis device, which comprises an analysis unit for measuring a component fed to the plurality of containers and analyzing the component thus measured, wherein the plurality of containers are at least a first container and a second container, wherein the first container retains a first solution containing a substance that releases adhesion between multiple cells forming the liver cell tissue, and the second container retains a buffer solution, and wherein the analysis unit measures an amount of the component discharged from the liver cell tissue in the first container into the first solution and an amount of the component discharged from the liver cell tissue in the second container into the buffer solution in the second container, and analyzes an amount of the component to be discharged via a bile duct in the liver cell tissue.

FIELD OF INVENTION

The present invention relates to an assessment in vitro (outside thebody) of an overall picture of pharmacokinetics, such as uptake,metabolism, and excretion, of a medicament, useful for new drugdevelopment.

BACKGROUND OF INVENTION

In the process of new drug development, a clinical trial (“humanclinical trial”) in which a drug is administered to human bodies toverify the efficacy is necessarily performed under the PharmaceuticalAffairs Act. However, a human clinical trial and an animal experimenttrial require an enormous development expense. In recent years, theentire cost for new drug development has increased due mainly to such adevelopment expense. One of main causes thereof is the fact that in thecase of a drug candidate whose short medical efficacy and whose toxicityare not able to be detected in non-clinical animal experiments in anearly phase of the development process, but found only in a humanclinical trial in a latter phase of the development process, theprevious development expenses and the expenses for the human clinicaltrial go to waste.

Against the background, in order to increase a pass rate of a humanclinical trial to reduce the cost for new drug development, it isimportant to screen a new drug candidate substance that has a medicalefficacy and does not show toxicity, in an early phase. Thus, manypharmaceutical companies demand an assessment system in vitro (outsidethe body) in which, in an early phase of a drug discovery, a drugcandidate is not screened only by an animal experiment which has a smallcorrelation with the characteristics of human cells, butpharmacokinetics of an administered medicament in human bodies can besuccessfully predicted using human cells.

However, a technique for grasping and dissecting an overall picture ofpharmacokinetics, such as uptake, metabolism, and bile duct and bloodvessel excretions, of an administered pharmaceutical candidate compoundby utilizing the cells has not been established yet. In order for a drugto exhibit a medical efficacy in the body, the drug once taken into aliver is required, after being converted into a metabolism product or ina form of the original compound (parent compound) as it is withoutundergoing metabolism, to be discharged into a blood vessel side andthen recirculate through the bloodstream again to reach a target organor part.

Accordingly, also in a trial system in vitro, if the recirculatingamount of a pharmaceutical candidate compound discharged to the bloodvessel side after administration can be measured to assess the medicalefficacy which is one of the most important indices in the new drugdevelopment, such a measurement can be a very useful assessment methodof pharmacokinetics. In addition, by grasping an overall picture ofpharmacokinetics including an amount of the compound excreted from thecells to the bile duct (elimination amount), which is then excreted outof the body with urine or feces after the bile duct excretion and anamount of the compound retained in the cells, it becomes possible todetermine a distribution ratio into fractions.

PTL 1 and PTL 2 have previously disclosed a technique in which a drug isadministered to cultured cells to assess an amount excreted from thebile duct of the cells (elimination amount). This assessment methodassesses a drug elimination amount which is an amount of theadministered drug that is excreted into the bile duct without exhibitingtoxicity or a medical efficacy, and then excreted out of the body withurine or feces.

CITATION LIST Patent Literature

PTL 1: Japanese Patent Publication No. 2012-65659

PTL 2: Japanese Patent Publication No. 8-503610

SUMMARY OF INVENTION Technical Problem

The prior art techniques were methods which assesses a bile ductexcretion amount, namely, an amount of a component that has no medicalefficacy, that is, methods for assessing the out-of-body-eliminationamount. However, in order to obtain information of a component having amedical efficacy, it is desired to directly assess the amount of thecomponent excreted to the blood vessel side. In the prior art, however,a method for analyzing an amount of a compound excreted into bloodvessels has not been established. Furthermore, there has not been anyestablished assessment method in which not only a part ofpharmacokinetics can be assessed, but also an overall picture ofpharmacokinetics in vitro can be provided to achieve a medical efficacyassessment with a higher accuracy, by dividing an administered drug intofractions and quantifying the fractions, such as a blood-vessel(basal/basolateral)-side discharge fraction, a lumen (apical)-sidedischarge fraction, and an intracellular retention fraction, todetermine where the administered drug is excreted from and where thedrug is retained in.

Solution to Problem

For example, provided is a component analysis device, including aretention unit for retaining a plurality of containers for retaining aliver cell tissue and an analysis unit for measuring a component fed tothe plurality of containers and analyzing the component thus measured,wherein the plurality of containers are at least a first container and asecond container, wherein the first container retains a first solutioncontaining a substance that releases adhesion between multiple cellsforming the liver cell tissue, and the second container retains a bufferliquid, and wherein the analysis unit measures an amount of thecomponent discharged from the liver cell tissue in the first containerinto the first solution and an amount of the component discharged fromthe liver cell tissue in the second container into the buffer liquid inthe second container, to analyze an amount of the component to bedischarged via a bile duct in the liver cell tissue.

Advantageous Effects of Invention

By applying the present invention, a component, such as a drug, excretedto the blood vessel side of the cells can be accessed in a directmanner. Furthermore, a blood-vessel (basal/basolateral)-side dischargefraction via a transporter and via diffusion, a lumen (apical)-sidedischarge fraction, and an intracellular retention fraction, of apharmaceutical candidate compound (parent compound and metabolismproduct) are quantified, and the total amount of the administeredpharmaceutical candidate compound and a distribution ratio intofractions are determined, whereby kinetics of the administeredpharmaceutical candidate compound can be assessed, an accuracy inscreening in vitro a drug candidate exhibiting a medical efficacy fromamong a huge number of pharmaceutical candidate compounds can beenhanced. As a result, it becomes possible to screen a pharmaceuticalcandidate compound in an early phase, and to reduce a wasted animalexperiment and an unnecessary human clinical trial. The presentinvention contributes to reduction of new drug development cost whichplaces a burden on pharmaceutical companies.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a culture plate.

FIG. 2 shows a sample preparation flow.

FIG. 3 shows objects to be quantified and medicament distribution imageviews for Steps 4, 5, and 6.

FIG. 4 shows a flow with 4-cell.

FIG. 5 shows a collection rates by a new protocol.

FIG. 6A shows fluorescence intensities of a collected drug in Testingsection 1, Step 5.

FIG. 6B shows fluorescence intensities of a collected drug in Testingsection 1, step 5.

FIG. 6C shows an effect of a dispersed drug.

FIG. 6D shows an effect of a dispersed drug.

FIG. 6E shows an effect of a dispersed drug.

FIG. 6F shows an effect of a dispersed drug.

FIG. 6G shows an effect of a dispersed drug.

FIG. 6H shows an effect of a dispersed drug.

FIG. 6I shows an effect of a dispersed drug.

FIG. 6J shows an effect of a dispersed drug.

FIG. 6K shows an effect of a dispersed drug.

FIG. 6L shows an effect of a dispersed drug.

FIG. 7 shows definitions of fractions and results for CDF.

FIG. 8A shows a distribution ratio of CDF.

FIG. 8B shows a distribution ratio of CDF.

FIG. 9A shows a distribution ratio of Rhodamine 123.

FIG. 9B shows a distribution ratio of Rhodamine 123.

FIG. 10 shows an automated measurement device configuration diagram.

FIG. 11 shows a sample preparation unit top view.

FIG. 12 shows a sample preparation unit front view.

FIG. 13A shows an automated measurement device operation flow chart.

FIG. 13B shows an automated measurement device operation flow chart.

FIG. 14 shows an automated measurement device operation flow chart.

FIG. 15A shows an automated measurement device operation flow chart.

FIG. 15B shows an automated measurement device operation flow chart.

FIG. 16A shows an automated measurement device operation flow chart.

FIG. 16B shows an automated measurement device operation flow chart.

FIG. 17 shows a chip head and a chip.

DESCRIPTION OF EMBODIMENT Example 1

In this example, a component analysis method for the medical efficacyassessment described above will be explained along Steps 0 to 6 in FIG.2. Steps 4 to 6 will be described in detail with reference to FIG. 3.Incidentally, in the following plural examples, although an explanationwill be made based on an assessment method of a medicinal efficaciousingredient, the method is merely an example for explaining the inventionof the present application, and the component analysis method of theinvention of this application can obviously be applied for assessing anyother chemicals than drugs, as well as medicament metabolism products.In addition, specific conditions, such as the buffer liquid,temperature, and time period, shown in this example, are merelyexamples, and other conditions may obviously be applied as long as theyhave the same effect in terms of a technical idea.

In this example, a case where at least three testing sections of TestingSections 1 to 3 are used is explained. In each testing section, aretention area for retaining cells are present, and in the retentionareas, respective containers may be used for respective testingsections, or one container having plural retention areas may bepartitioned to set the respective testing sections.

<Step 0: Preparation and Culture of Liver Cells>

In this step, liver cells are prepared and cultured for verifying aneffect of a drug. An example will be shown below. The preparation ofliver cells was made according to an in-situ collagenase perfusionmethod. The detail is as follows. A rat (5 to 6 weeks old) is subjectedto laparotomy under pentobarbital anesthesia, and a catheter is insertedinto the portal vein and a pre-perfusion fluid (Hanks' solution notcontaining Ca2+ and Mg2+ but containing EGTA) is injected.

After thorough blood removal from the liver is confirmed, the perfusionis stopped. The perfusion fluid is replaced with a collagenase solution,and then perfusion is performed. In this example, perfusion is performedusing a Hanks' solution containing 0.05% collagenase, but the perfusionfluid is not limited thereto. After digestion of the intercellulartissue by collagenase is confirmed, the perfusion is stopped. The liveris cut out, cut into elongated segments in a cooled Hanks' solution, anddispersed as cells by pipetting. Damaged liver cells are removed bycentrifugation of 500 G for 5 minutes using isotonic Percoll.

The viability of the resulting liver cells is measured by the trypanblue exclusion method, liver cells of a viability of 85% or more areused for culture. Although liver cells of a viability of 85% or more areused for culture here, the present invention is obviously not limited tothe condition. In addition, preparation of liver cells is notnecessarily limited to the in-situ collagenase perfusion method. Theliver cells to be used are not limited to those originated in a rat, andthe lineage of the rat is not limited. Although liver cells were used inthis example, the present invention is not limited thereto.

Liver cells prepared by the in-situ collagenase perfusion method asdescribed above are suspended in a medium, and the liver cells in thesuspension at a density of 5×10⁵ cells/mL are seeded on a commerciallyavailable collagen-coated culture dish, to conduct two dimensional planeculture (for example, sandwich culture). The seeding density, medium,culture plate 001 are not particularly limited.

The culture plate is shown in FIG. 1. Although a 24-well culture platecontaining 24 culture areas (wells, 002) is illustrated here, theculture plate is not limited thereto and a container having anothershape may be used as long as given cells can be retained therein.

After seeding, culture is started using a CO2 incubator under conditionsof 5% CO2 and 37° C. After 18 hours or more elapse, a first mediumexchange is performed. Although a medium used for culture from 18 hoursafter the seeding is not particularly limited, in this example, a mediumobtained by excluding FCS from a medium (10% FCS+) to make a medium(herein after medium (FCS−)) and then adding matrigel thereto was used.After that, medium exchange is performed with the medium (FCS−) every 24hours.

In addition, in a case where cells are cultured in a three-dimensionalspheroid, that is, in this example, in a case where athree-dimensionally formed liver cell tissue is cultured, a 24-wellnanopillar cell culture plate may be used. The number of wells is notparticularly limited.

The cells prepared at a density of 5×10⁵ cells/mL are seeded, and thenculture is started using a CO2 incubator under conditions of 5% CO2 and37° C. After 18 hours or more, a first medium exchange is performed.Although a medium used for culture from 18 hours after the seeding isnot particularly limited, in this example, a medium obtained byexcluding FCS from a medium (10% FCS+) (hereinafter, referred to asmedium (FCS−)) was used.

Although a medium used for culture from 48 hours after the seeding isnot particularly limited, in this example, a medium obtained byexcluding FCS from a medium (10% FCS+) to make a medium (herein aftermedium (FCS−)) and then adding matrigel thereto was used. After that,medium exchange is performed with the medium (FCS−) every 24 hours.

As described later, since tests under different three types ofconditions are conducted in Step 5 (described in detail in Testingsections 1, 2, and 3: Step 5), three culture plates having the samecondition are independently prepared at this point. In Steps 0 to 4 andStep 5, the same testing operation is conducted for three types oftesting sections.

<Step 1: Conditioning of Liver Cells>

In this step, cells cultured in Step 0 are conditioned to a conditionsuitable for a drug assessment. An example will be shown below. Theculture supernatant of cells cultured for 4 days in Step 0 is removed,400·L of a Hanks' solution is added as a buffer, and then incubation isperformed at 37° C. for 10 minutes (FIG. 2, Step 1).

The kinds and amount of the buffer is not particularly limited. Theoperation of Step 1 is desirably repeated twice. When a component inwhich the cells settle is replaced from a medium used in culture in Step0 into a buffer liquid (for example, Hanks' solution) in this manner, agroundwork can be prepared for conducting accurate measurement anddissection in the subsequent steps. However, the number of repetitionsof Step 1 may obviously be arbitrarily changed depending on the kinds ofthe buffer liquid and the cells used.

<Step 2: Drug Solution Administration>

In this step, a drug solution to be assessed is administered to cells.An example will be shown below. After removing the buffer, 200 μL of 10μM CDF (fluorescent reagent) is added to a well, incubation is performedat 37° C. for 30 minutes, the well is then retained at 4° C. for 5minutes (FIG. 2, Step 2).

The kind, concentration, and amount of the reagent are not particularlylimited. CDF emits fluorescence, and therefore can be easily quantifiedby a plate reader as a model reagent. In addition, the administeredamount, which was 200 μL, was selected as such an amount that allows forall the cells in the well to be immersed in the reagent. The amount isnot limited to such an amount as long as all the cells are immersed inthe reagent. The CDF concentration may be any concentration that hasbeen heretofore used in a fluorescent assay of a cell. The aboveconcentration is desirably applied in terms of the amount of reagent fordetection with a plate reader. The time period of the incubation, whichwas 30 minutes, was adopted based on a result of a previous study inwhich a time period for reaching a significant equilibrium condition inthe drug intake-discharge rate was 30 minutes. The time period is notlimited but depends on the purpose, and plural time periods may becombined. In addition, incubation is desirably performed using a CO2incubator under conditions of 5% CO2 and 37° C., as with the case ofStep 0.

For the purpose of preventing the administered drug from leaking outsidethe cells, after the incubation for 30 minutes, the plate temperaturewas lowered to 4° C. This is conducted for causing a phenomenon that acell suppresses discharge of a drug to the outside when the temperatureinside the container is lowered.

By lowering the temperature of the drug to a prescribed temperature orlower in this manner, the drug remains in the cells, and the phenomenonof the drug leaking out of the cells can be suppressed. The temperature,which was 4° C. in this example, is not limited thereto as long asleaking of the administered drug out of the cells can be suppressed.

In addition, the method for suppressing leaking of the administered drugout of the cells is not limited to the method of lowering thetemperature if another method, for example, a method of administering aninhibitor, can be applied. In any methods, the temperature forpreventing discharge or leaking of the administered drug out of cells islower than the plate temperature upon administering the drug solution(for example, 37° C.). It is because uptake of the administered drugsolution into cells is a biological phenomenon, which is considered tobe typically activated at 37° C.

The timing of the drug administration is not limited to this example.For example, the drug administration is sometimes started on the daybefore the test day or several days earlier. In this case, the drug maybe administered at Step 0 to omit Steps 1 and 2.

<Step 3: Cell Washing>

In this step, any other substances than the drug solution retained inthe cells are washed off by Step 3. An example will be explained below.Next, while holding the plate at 4° C., cells are washed with 400 μL ofan ice-cooled Hanks' solution 3 times (FIG. 2, Step 3).

The reason of 4° C. is the same as described above. The amount of themedium in culturing was 400 μL, and for the purpose of removing a mediumcomponent remaining on the inside wall of the well, the amount of thewashing Hanks' solution was set to 400 μL. The number of times ofwashing in an ordinary biochemical assay, which is typically three, wasfollowed here. The conditions of washing amount, number of times ofwashing, or the like are not limited. In this manner, it is possible toremove any other substances than the drug solution to be measured toenhance the accuracy of the medical efficacy assessment in thesubsequent steps.

<Step 4: Blood-Vessel-Side Discharge Fraction Collection>

As one index for assessing a medical efficacy, it is effective toanalyze whether an administered drug is likely to remain in cells for along period of time or for only a short period. Thus, in this step, inorder to acquire data for the index, prior to an assessment of cells ineach testing section in Step 5 described later, the drug is leaked fromcells for a predetermined time. An example will be shown below. First, abuffer liquid for a pretreatment (for example, Hanks' solution) wasadministered, incubation was performed at 37° C. for 30 minutes, and theHanks' solution containing the drug (supernatant) was collected (FIG. 2,step 4).

The incubation time is defined depending on the used drug and thepurpose, and is not limited to 30 minutes as in this Example. Inaddition, the incubation is desirably performed using a CO2 incubatorunder conditions of 5% CO2 and 37° C., as with the case of Step 0. Asfor the buffer liquid, a buffer liquid of the same kind as one used instep 5 may be used, or one of other kinds may be used.

In order to discharge the drug that had been retained in the liver celltissue (FIG. 3, 101) until Step 3 into the Hanks' solution through firstblood-vessel-side discharge, that is, via passive diffusion (FIG. 2,Step 4, (1)′) and via a transporter (TP) (FIG. 2, Step 4, (2)′ and 102in the image view), the temperature was maintained at 37° C. Asdescribed later, since blood-vessel-side discharge is performed also inStep 5, for discriminating them, the blood-vessel-side discharge in Step4 was defined as the first blood-vessel-side discharge, and theblood-vessel-side discharge in Step 5 was defined as a secondblood-vessel-side discharge.

Essentially, both of the steps are a step for discharge from cells tothe blood vessel side (basal/basolateral) (supernatant). Alternatively,depending on the purpose, Step 4 can be considered as a washing stepprior to the collection step by Step 5. A transporter is a membraneprotein that is expressed on a cell membrane and has a function totransport a substance. A transporter plays a role of active substancetransportation between the inside and the outside of a cell.

In addition, the passive diffusion is discharge out of the cell notthrough a transporter, including leaking from a cell membrane, or thelike. The reason why the drug discharged into the Hanks' solution isdefined as the blood-vessel-side discharge fraction in step 4 is thatthe top surface portion of the cell facing the Hanks' solutioncorresponds to a basal/basolateral surface (FIG. 3, 104), which isassumed to be a part facing a blood vessel in the body.

Step 5 explained below is a step for collecting the drug by different 3kinds of operations (Testing sections 1, 2, and 3). By obtaining, priorto step 5, the first blood-vessel-side discharge fraction (a drugdischarged into the Hanks' solution) in advance in Step 4 as describedabove, an index for verifying whether or not the drug is apt to remainin the cell can be acquired.

Incidentally, as the Hanks' solution used for grasping theblood-vessel-side discharge fraction, the Hanks' solution of any testingsection may be used. For example, all the Hanks' solutions for Testingsections 1 to 3 may be assessed or only a part thereof may be used. Inaddition, Steps 3 and 4 are steps for performing the analysis of thepresent invention in more detail to achieve a highly accurateassessment, and these steps may obviously be skipped to perform anoperation of Step 5.

Steps 0 to 4 described above are, as shown in FIG. 2, performed at leastin Testing sections 1 to 3. In Step 5, in order to acquire plural indexdata to be determined in the present invention by analysis describedlater, in each of Testing sections 1 to 3, a treatment under a differentcondition is performed. An example of Step 5 will be shown below.

On the other hand, for quantifying the total amount of the drugadministered to cells, some other cells than the cells used in Testingsections 1 to 3 are separately provided in advance, and a step forbreaking the cells and collecting a cell extract solution was addedbetween Step 3 and Step 4 (Step 4.0, FIG. 4).

A fraction obtained in this step is referred to as a 4-cell fraction.The 4-cell fraction corresponds to a container which is separatelyprovided from the containers used in Testing sections 1 to 3, that iscorresponds to a sort of Testing section 4. By adding this step, it ispossible to determine the collection rate of the drug from the sum ofthe drug amounts collected in Testing sections 1 to 3 in the subsequentSteps 4, 5, and 6 relative to the total drug amount obtained in Testingsection 4. By determining the collection rate of the drug, it ispossible to assess the drug intake-discharge rate.

In the case of a fluorescence measurement, for example, either in a 2Dstructure such as sandwich culture or in a 3D structure such asspheroid, the drug collection and measurement of the fluorescenceintensity can be performed by following the collection protocol forfluorescence measurement shown in FIG. 2, Step 6.

In addition, also in an LCMS measurement, for example, in the case of a2D structure such as sandwich culture, the drug collection and LCMSmeasurement can be performed by following the collection protocol forLCMS measurement shown in FIG. 2, Step 6.

On the other hand, in the case of a 3D structure such as spheroid,collection of the whole amount of the drug has been difficult even withthe collection protocol for LCMS measurement shown in FIG. 2, Step 6.Accordingly, a protocol in which trypsin/EDTA is added as described inStep 4.0 in FIG. 4 has been devised.

This aims at cutting an intercellular adhered protein by trypsin whichis a proteinase to degrade spheroid into single cells, facilitatingaccess with water and methanol, and thereby extracting the intracellularfraction efficiently.

The reagent is not limited to this reagent as long as it can releaseintercellular adhesion in a structure in which cells are threedimensionally arranged such as spheroid to degrade the structure intothe single cells. In addition, the reagent may be any reagent that canextract the intracellular fraction in a 3D structure even not throughdegrading the structure into the single cells, and can be applied to aliquid chromatography mass spectrometry (LCMS) measurement. An examplein which 10 M of Rosuvastatin is administered to cells and a LCMSmeasurement is performed to assess a drug intake-discharge rate is shownin FIG. 5.

The drug intake-discharge rate can be calculated by (Steps4+5+6)/4-Cell×100. As a result, a collection rate in the case where a 2Dstructure was subjected to a water and methanol treatment was 92.3%, anda collection rate in the case where the 2D structure was subjected to atrypsin treatment, followed by a water and methanol treatment was 94.0%.

On the other hand, a collection rate in the case where a 3D spheroid wassubjected to the water and methanol treatment was 138%, and a collectionrate in the case where the 3D spheroid was subjected to the trypsintreatment, followed by the water and methanol treatment was 92.2%. Thereason why the collection rate in the case of a 3D spheroid treated onlywith water and methanol exceeded 100% was considered as follows: thedrug of the 4-cell fraction was not be able to be fully collected onlywith water and methanol and thus the sum of the drug amounts collectedin the subsequent Steps 4, 5, and 6 exceeded the drug amount of the4-cell fraction. This suggests that a trypsin treatment degradesspheroid into the single cells to facilitate access of water andmethanol, and allows the intracellular fraction to be extractedefficiently.

As is apparent from the above results, in order to collect theintracellular fraction completely to obtain an precise drugintake-discharge rate, a trypsin treatment is not necessary in a 2Dstructure, but essential in a 3D spheroid. The reagent is obviously notlimited to trypsin/EDTA as long as it is a reagent that releasesintercellular adhesion to facilitate degradation into the single cells.

<Step 5, Testing Section 1: Collection of Supernatant Through 37°C.-Collapsed System>

In Testing section 1, as illustrated in Testing section 1 of FIG. 3, thetotal of the bile-duct-side discharge (3) and the secondblood-vessel-side discharge (that is, discharge by diffusion (1) and atransporter (TP) (2)) is discharged from cells into a buffer liquid (forexample, Hanks' (−) solution).

The Hanks' (−) solution is a Hanks' solution containing no calcium ionand no magnesium ion, and used in the case, such as this testingsection, where intercellular adhesion is intentionally not to bestrengthened. In the case of a sandwich culture with a two dimensionalplane form, for the purpose of actively releasing intercellularadhesion, a chelator such as EGTA as described later is used.

Next, 200 μL of the Hanks' (−) solution containing 1 mM of EGTA wasadded. EGTA has a chelating function for suppressing an action of Ca2+and Mg2+ which involve in adhesion of intercellular adhesion molecules,and is a reagent for releasing the intercellular adhesion. This allows abile duct formed in the culture process to collapse.

After incubation at 37° C. for 30 minutes, the supernatant wascollected. The incubation is desirably performed using a CO2 incubatorunder conditions of 5% CO2 and 37° C. as with the case of Step 0. Thereagent is not limited to EGTA as long as it has a chelating function.The kind and amount of the buffer containing EGTA and the temperatureand time period for the incubation are not particularly limited.

In Testing section 1 of Step 5, at the time when Step 4 is ended, all ofthe drug retained in the cells (FIG. 3, Step 5, and image views (1) and(2)) and the drug discharged into the bile duct (FIG. 3, Step 5 andimage view (3)) are discharged into the supernatant and collected (FIG.3, Step 5, Testing section 1).

The reason why (3) is defined as a bile-duct-side discharge fraction inStep 5 is that a cell membrane part around a gap formed in theintercellular adhesion part corresponds to an apical face (FIG. 3, 105),and that the gap is assumed as a bile canaliculi (FIG. 3, 103).

In Step 5, Testing section 1, the temperature is maintained at 37° C.,and therefore a fraction discharged from cells into the blood vesselside includes a fraction via passive diffusion (FIG. 3, Step 5, (1)) anda fraction via a transporter (FIG. 3, Step 5, (2)). In addition, in thecase of 3D spheroid culture, since the cells formed several layers orfor other reasons, even when the treatment with a buffer containing EGTAwas performed for a appropriate time period for a two dimensionalsandwich culture, the intercellular adhesion release did not proceed andit was difficult to collect the bile duct discharge fraction (FIG. 6A).

For this reason, for a 3D spheroid obtained after adding 200 μL of 10 μMof CDF (fluorescent reagent) to a well in Step 2 and performingincubation at 37° C. for 30 minutes, trypsin/EDTA was used in Step 5,Testing section 1.

This was performed for the purpose as follows: since it was considereddifficult for only EGTA to release the intercellular adhesion of the 3Dstructure composed of several layers of cells within an appropriate timeperiod, the intercellular adhesion protein is more actively cut into thesingle cells with trypsin which is a protease.

The detail of the protocol is as described in FIG. 4, Testing section 1,Step 5. Results of fluorescence intensity measurements of thesupernatant, taken immediately after the buffer administration, and at 2minutes, 5 minutes, 10 minutes, and 20 minutes after administration areshown in FIG. 6B. The case of only the buffer is plotted by ▴, the caseof EGTA by ▪, the case of trypsin/EDTA by ●. As a result, it was foundthat, unlike in the case of treatment with only EGTA (▪), in the case oftreatment with trypsin/EDTA (●), the supernatant-side discharge amountapparently increased (FIG. 6B).

The reason was considered as follows: as is apparent from the phasecontrast microscopic image in FIGS. 6C to 6L, intercellular adhesion wascollapsed by the trypsin/EDTA treatment, and the bile duct dischargefraction was discharged to the supernatant side. This suggests that forcompletely collecting the bile duct discharge fraction from a 3Dspheroid, a treatment for degrading the cells into the single cells isessential. The reagent is obviously not limited to trypsin/EDTA as longas it releases intercellular adhesion to facilitate the degradation intosingle cells.

<Step 5, Testing Section 2: Collection of Supernatant Through 37°C.-Maintained System>

In Testing section 2, the bile duct is not collapsed unlike in Testingsection 1 and only a fraction that is called the secondblood-vessel-side discharge in FIG. 3 (diffusion (1) and transporter(2)) is discharged. The Hanks' solution was added in an amount of 200μL. Since Testing section 2 does not contain a chelating agent such asEGTA unlike Testing section 1, intercellular adhesion remains to bemaintained, and therefore, in this testing section, collapse of a bilecanaliculi is not induced.

After incubation at 37° C. for 30 minutes, the supernatant wascollected. The incubation is desirably performed using a CO2 incubatorunder conditions of 5% CO2 and 37° C. as with the case of Step 0.

In Testing section 2 of Step 5, the drug retained in the cells at thetime when Step 4 ends (FIG. 3, Step 5, and image view (1), (2)) isdischarged into the supernatant and collected (FIG. 3, Step 5, Testingsection 2). Various conditions are not limited.

According to the foregoing explanations, the drug discharged in thesupernatant is defined as the second blood-vessel-side dischargefraction. In Step 5, Testing section 2, since the temperature ismaintained at 37° C., a fraction discharged from cells into the bloodvessel side includes a fraction via passive diffusion (FIG. 3, Step 5,(1)), and a fraction via a transporter (FIG. 3, Step 5, (2)).

By quantitatively analyzing amounts of the drug collected from Testingsection 1 and Testing section 2 of Step 5, for example, by subtracting aquantified value of the amount of the drug collected in Testing section2 from a quantified value of the amount of the drug collected in Testingsection 1, a drug amount of the bile-duct-side discharge (3) can becalculated.

<Step 5, Testing Section 3: Collection of Supernatant Through 4°C.-Maintained System>

By making Testing section 3 in a lower temperature condition thanTesting sections 1 and 2 as described later, as for the secondblood-vessel-side discharge, the discharge of the drug from atransporter (1) is suppressed, and only the discharge fraction throughdiffusion (2) is discharged. After 200 μL of a Hanks' solution was addedand incubation was performed at 4° C. for 30 minutes, the supernatantwas collected. This testing section is different in the test temperaturefrom Step 5, Testing section 2 that shares a common point of amaintained system.

This is because the blood-vessel-side discharge via a transporter issuppressed by adopting 4° C. In Step 5, Testing section 3, by adoptingTesting section of a low temperature, 4° C. in which a transporteractivity is suppressed, it is possible to quantify only a dischargeamount via passive diffusion (FIG. 3, Step 5, (1)) to calculate(quantify) the blood-vessel-side drug discharge amount via a transporterin comparison with Step 5, Testing section 2.

For each drug, a specific transporter is present for moving inside andoutside the cells. Accordingly, the possibility of excluding thedischarge amount via passive diffusion and quantifying discharge amountvia a transporter means possibility of quantifying a specific dischargeamount for each drug. The kind and amount of the buffer, and thetemperature and time period of incubation are not particularly limited.

By quantifying the drug collected from Testing section 2 and Testingsection 3 of Step 5 in the same manner as described above, the dischargefraction via a transporter can be calculated. Incidentally, an examplein which Testing sections 1, 2, and 3 are used to calculate therespective discharge fractions was described in this example, but thenumber of the testing sections is not necessarily limited to three.

For example, if only the bile-duct-side discharge (3) and the secondblood-vessel-side discharge ((1)+(2)) are desired to be separated, onlyTesting sections 1 and 2 have to be conducted, and if merely thediffusion (1) and the transporter (2) are desired to be separated in thesecond blood-vessel-side discharge, only Testing sections 2 and 3 haveto be conducted.

In addition, by separately providing a 4° C.-collapsed system as needed,as for the discharge into the bile-canaliculi-side, discharge via atransporter is suppressed, and only the discharge by diffusion can bemeasured. This makes it possible to separately assess the discharge intothe bile canaliculi side by diffusion and the discharge into the bilecanaliculi side via a transporter, for example, by comparing the 4°C.-collapsed system with Testing section 3. The steps in each of Testingsections 1 to 3 described above may be conducted in any order, or thesteps may be conducted in parallel.

<Step 6: Collection of Bile Duct Fraction and Intracellular Fraction>

In Step 6, all the three testing sections return into a common operationagain. In Step 6, drug collection is performed for quantifying theintracellular retention fraction. In the case where a fluorescence drugor the like is quantified with a plate reader or the like, it is desiredthat a Hanks' solution containing 1% of a surfactant is added, forexample, in an amount of 200 μL, to suspend the cells therein, and thewhole amount is collected (FIG. 2, Step 6).

This makes it possible to break a cell membrane to discharge the drugremaining in the cells. The resulting sample was transferred into aculture plate, and a fluorescence measurement was performed using aplate reader. For a blank measurement, a well in which only a Hanks'solution is added is provided. The fluorescence intensity is measuredusing an excitation wavelength of 484 nm and an absorption wavelength of519 nm.

In addition, when a drug is quantified using a mass spectrometry (LCMS)device, or the like, the drug remaining in cells is extracted throughmaking a hypotonic solution by adding water, a treatment with an organicsolvent, or other procedure, and then, the cells are suspended in anorganic solvent such as methanol to collect the whole amount (FIG. 2,Step 6).

Subsequently, the drug is quantified using a LCMS device as describedlater. Either in the case of a plate reader measurement of afluorescence drug as described above, or in the case of a LCMSmeasurement of a drug, when a 3D spheroid is used as a material, a cellpellet collected after centrifugation in Testing section 1, Step 5 issubjected to the breaking step described above and the drug isquantified.

<Determination of Distribution Ratio into Fractions and Scoring Based onMeasurement Results>

Based on fluorescence intensities reflecting the drug amounts obtainedin the above steps, a distribution ratio into fractions is calculated.Here, the total sum of Steps 5 and 6 (FIG. 3, B #+C #=(1)+(2)+(3)+(4))is taken as a 100% drug amount.

This case is referred to as “Pattern 1”. From 6 kinds of valuesdetermined by the fluorescence amount measurements (B1, B2, B3, C1, C2,and C3), a distribution ratio into fractions as shown in FIG. 7, Pattern1 is obtained. From the values,

the second-blood-vessel-side discharge fraction only by diffusion(extracellular efflux by diffusion, ExEfx-Dif) is associated to B3,

the second-blood-vessel-side discharge fraction only via a transporter(extracellular efflux by transporter, ExEfx-TP) is associated to B2-B3,

the bile-duct-side discharge fraction (bile canaliculi efflux, BCEfx) isassociated to B1-B2,

the intracellular retention fraction (Cell) is associated to C1.

Based on the results, a circular graph, such as the graph of Pattern 1in FIG. 8A, can be drawn, making it possible to visually understand anoverall picture of the distribution ratio into fractions. Furthermore,based on the quantification results, it is possible to make a scoringpeculiar for each drug as described below.

In this example, an example of a CDF score calculated will be shown. Thescore calculated is not limited thereto. A score for assessing ablood-vessel-side discharge via a transporter is obtained by

(B2−B3)/B2 as a ratio of a drug amount discharged via a transporterrelative to the second blood-vessel-side discharge drug amount (ratio ofextracellular efflux by diffusion, RexEMTP). A score for assessingexcretion into a bile duct is obtained by

(B1−B2)/(C1+C2) as a ratio of a bile duct excretion drug amount relativeto the whole drug amount taken in cells (biliary retention drug, BiRD),

or, alternatively, obtained by

(C2−C1)/C2, (B1−B2)/C1, or the like, as a ratio of a bile duct excretiondrug amount relative to a drug amount remaining in cells.

<Comparison of Distribution Ratios and Scores Between Different Drugs>

By replacing CDF with Rhodamine 123, results shown in FIG. 9A(Pattern 1) can be obtained in the same operation as described above. Itcan be seen that a clearly different distribution ratio can be detectedfrom FIG. 8A (Pattern 1) which shows results for CDF.

In addition, for example, it can be seen that the ratio of the drugamount discharged via a transporter relative to the blood-vessel-sidedischarge drug amount of CDF (RexEMTP) and the ratio of the bile ductexcretion drug amount relative to the whole drug amount taken in thecells (BiRD) are 41.08 and 18.58, respectively, whereas those ofRhodamine 123 are 52.52 and 4.92, respectively. Thus, it can be seenthat CDF is a drug that has a property of being more likely to beexcreted into a bile duct and less likely to be discharged to the bloodvessel side, as compared with Rhodamine 123. As described above,according to the present invention, it is possible to assess whatpharmacokinetics each compound exhibits.

Example 2

In Example 2, determination of a distribution ratio into fractions andscoring based on measurement results will be explained in the case ofusing a different method from that in Example 1. In this example, it ispossible to give, in addition to the distribution ratio into fractionsshown in Pattern 1 of Example 1, information on whether or not theadministered medicament is likely to remain in cells by utilizing thequantified values S # (corresponding to Step 4) shown in FIG. 3, and toachieve a more accurate assessment.

Specifically, the total sum of Step 4, 5, and 6 (FIG. 3, S #+B #+C#=(1)′+(2)′+(1)+(2)+(3)+(4)) is taken as a 100% amount of theadministered medicament. This case is referred to as “Pattern 2”.Accordingly, the 9 kinds (S1, S2, S3, B1, B2, B3, C1, C2, and C3)determined by the testing sections of Step 4, 5, and 6 can be used asindices for assessment.

From the 9 kinds of values determined by the fluorescence amountmeasurements, the distribution ratio into the fractions as shown inPattern 2 of FIG. 7 can be obtained. From the values,

the first blood-vessel-side discharge fraction (Sup fraction) isassociated to S1 (≅S2≅S3),

the second blood-vessel-side discharge fraction only by diffusion(extracellular efflux by diffusion, ExEfx-Dif) is associated to B3,

the second blood-vessel-side discharge fraction only via a transporter(extracellular efflux by transporter, ExEfx-TP) is associated to B2−B3,

the bile-duct-side discharge fraction (bile canaliculi efflux, BCEfx) isassociated to B1−B2, and

the intracellular retention fraction (Cell) is associated to C1.

Based on the above results, a circular graph as shown in Pattern 2 ofFIG. 8B can be drawn, making it possible to visually understand anoverall picture of the distribution ratio into fractions. With themethods shown in Example 2, the sup fraction which is an index forwhether or not the medicament is likely to remain in the cells is 70.82for CDF, but 39.29 for Rhodamine 123 (FIG. 9B, Pattern 2), and it can beseen that CDF tends to be discharged to the blood vessel side.

This matches the assessment of the intracellular retention fraction inExample 1. Furthermore, based on the quantification results, a scoringpeculiar for the drug can be made, which is as shown in Example 1.

Example 3

In Example 3, an example of a device for realizing automation of aseries of steps described in Example 1 and 2 will be described.Incidentally, descriptions of the purpose of the operation of the devicedescribed below, the role of the component thereof, and the like may besometimes omitted when they are the same as in Example 1 or 2.

In the device configuration described below, the areas for retainingcells set for the respective Testing sections 1 to 3 shown in Examples 1to 2 are explained with expressions of a “well for Testing section 1”,“well for Testing section 2”, “well for Testing section 3”, and thelike. As for the areas, for example, a plurality of containers each ofwhich is the aforementioned “well culture plate” are set for therespective testing sections, or a plurality of wells in one well cultureplate may be partitioned to set the partitioned areas as, for example,“a first container”, “a second container”, “a third container”, and thelike. Incidentally, in this case, the 4-cell fraction (Testing section4) explained in Example 1 described above is another “fourth container”that is different from the first to third containers.

In particular, since the well for Testing section 1 and the well forTesting section 2 are adjusted to almost the same temperature, a moreefficient assessment can be achieved in terms of the accuracy and speedwhen the sections are set by partitioning one well culture plate.

Incidentally, in the operation of the device described later, as for anoperation for replacing a well (container) for each testing section, thewell may be replaced automatically or by hand. The replacement andinstall operations of the containers are omitted in the followingexplanation.

As shown in FIG. 10, this device is composed of a culturing unit 106, asample preparation unit 107 (including an input unit 107A), an analysisunit 108, and a display unit 109. Furthermore, the sample preparationunit 107 includes a temperature regulation unit 107B for regulatingtemperatures inside containers (plate(s)) described later, a liquidfeeding unit 107C capable of feeding or collecting a liquid to or fromthe containers, and the like. The sample preparation unit is desirablyunder an environment of 5% CO2 and 37° C.

The analysis unit 108 includes a measurement unit 108A for measuringamounts of a component such as a drug and an analyzing unit 108B foranalyzing the amounts of fractions discharged via a transporter and viaa bile duct, remaining in cells, and discharged from other paths thanthe transporter and the bile duct (diffusion) from the amounts of thecomponent such as a drug obtained by the measurement unit. The deviceconfiguration is an example, and, for example, a configuration in whichthe analyzing unit is implemented by another device, to whichinformation obtained by the measurement unit is sent, may be obviouslyemployed.

In addition, the detailed configuration of the sample preparation unitis illustrated in FIGS. 11 and 12. As described above in Examples 1 and2, the sample preparation unit has a role of automatically preparing thefractions to be analyzed.

Each component will be explained with reference to flowcharts describedlater. An automated measurement device operation flow chart is shown inFIG. 13. The flowchart of FIG. 13 is merely an example, and as describedin Example 1, <step 2>, in the case where the drug is administered at adifferent time, the flowchart is not limited thereto. In this example, aplate including multiple cell retention areas (wells) is used as anexample of a container for retaining cells, but the container isobviously not limited to a plate as long as it can retain cells.

<Transfer from Culturing Unit to Sample Preparation Unit>

First, a liver cell tissue is cultured in the culturing unit 106 (FIG.10, FIG. 13A, Sub-step 1). Then, the plate retaining the cultured livercell tissue is transferred onto a firsttemperature-regulating-function-equipped plate holder 210 and a secondtemperature-regulating-function-equipped plate holder (211) of a samplepreparation unit (FIG. 10, FIG. 13A, Sub-step 2).

<Sample Preparation: Corresponding to Step 1 of Examples 1 and 2>

A suction head 205 which is placed in the liquid feeding unit 107C andprovided with a suction nozzle for sucking a liquid is moved to a well002 on a culture plate 001 filled with a medium to be removed, on theplate holder, and sucks the medium from the well to remove the wholeamount (FIG. 13A, Sub-step 3). The medium removed is collected fordisposal in a waste liquid tank 206.

Next, a chip 302 for retaining a liquid 301 is attached to a chip head204 mounted on the suction nozzle from a chip rack 207 storing aplurality of chips. The chip head moves to a normal temperature drugsolution rack 209, and sucks a buffer (FIG. 13A, Sub-step 4). The chiphead moves to a target well, adds the buffer (FIG. 13A, Sub-step 5), andthen moves to a dust box 214 to discard the chip. Although replaceablechips are used here for preventing contamination, the present inventionis not limited thereto.

The suction head moves to the well filled with the buffer and removesthe buffer (FIG. 13A, Sub-step 6). The removed medium is collected fordisposal into the waste liquid tank 206. This step is repeated twice intotal (washing step) (FIG. 13A, Sub-step 7).

Next, a chip in the chip rack 207 is attached to the chip head 204, andthe chip head moves to the normal temperature drug solution rack 209 andsucks the buffer (FIG. 13A, Sub-step 8). The chip head moves to thetarget well, adds the buffer, and then moves to the dust box 214 todiscard the chip. The device waits at 37° C. for 10 minutes(conditioning) (FIG. 13A, Sub-step 9). Then, the suction head 205 movesto the well filled with the buffer, and removes the whole amount of thebuffer (FIG. 13A, Sub-step 10).

<Sample Preparation: Corresponding to Step 2 in Examples 1 and 2>

A chip in the chip rack 207 is attached to the chip head 204, and thechip head moves to the normal temperature drug solution rack 209, andsucks a drug solution (FIG. 13A, Sub-step 11). The chip head 204 movesto the target well, adds the drug solution (FIG. 13A, Sub-step 12), andthen moves to the dust box 214 to discard the chip. The device waits at37° C. for 30 minutes (FIG. 13A, Sub-step 12).

Then, the first temperature-regulating-function-equipped plate holder210 and the second temperature-regulating-function-equipped plate holder211, which constitute one configuration example of the containerretention unit for retaining containers, change from 37° C. to 4° C.(FIG. 13A, Sub-step 13), then the suction head 205 moves to the wellfilled with the drug solution, and removes the whole amount of the drugsolution (FIG. 13A, Sub-step 14). Incidentally, as the temperatureregulation unit in this example, a plate holder is equipped with atemperature regulating function in this explanation, but the temperatureregulation unit may obviously be present separately from the containerretention unit.

<Sample Preparation: Corresponding to Step 3 in Examples 1 and 2>

A chip in the chip rack 207 is attached to the chip head 204, and thechip head moves to the cold-stored drug solution rack 208 and sucks abuffer (FIG. 13A, Sub-step 15). The reason why a cold-stored drugsolution is used is to stop an active biological phenomenon such as atransporter activity, as described above.

The cold-stored drug solution rack 208 is intended for keeping a drugsolution, a buffer, or the like at a low temperature for this purpose.The chip head 204 moves to the target well, adds the buffer thereto(FIG. 13A, Sub-step 16), and then moves to the dust box 214 to discardthe chip.

The suction head moves to he well filled with the buffer, and removesthe buffer (FIG. 13A, Sub-step 17). The removed medium is discarded intothe waste liquid tank 206. This step is repeated 3 times in total(washing step) (FIG. 13A, Sub-step 18).

<Sample Preparation: Corresponding to Step 4 in Examples 1 and 2>

After the first temperature-regulating-function-equipped plate holder210 and the second temperature-regulating-function-equipped plate holder211 change from 4° C. to 37° C. (FIG. 13A, Sub-step 19), a chip in thechip rack 207 is attached to the chip head 204, the chip head moves tothe cold-stored drug solution rack 208, and sucks the buffer (FIG. 13A,Sub-step 20). The chip head 204 moves to the target well, adds thebuffer (FIG. 13A, Sub-step 21), and then moves to the dust box 214 todiscard the chip.

The device waits at 37° C. for 30 minutes (FIG. 13A, Sub-step 20). Then,the first temperature-regulating-function-equipped plate holder 210 andthe second temperature-regulating-function-equipped plate holder 211change from 37° C. to 4° C. (FIG. 13A, Sub-step 22). Then, a chip in thechip rack 207 is attached to the chip head 204, moves to the well filledwith the buffer containing the drug, sucks the drug-containing buffer(supernatant), and dispenses the supernatant to a collection plate forcollection on the first plate holder 212 and the second plate holder 213(collection) (FIG. 13A, Sub-step 23).

<Sample Preparation: Corresponding to 4-Cell Fraction Preparation inExamples 1 and 2>

[For 2D Tissue]

After the first temperature-regulating-function-equipped plate holder210 and the second temperature-regulating-function-equipped plate holder211 change from 4° C. to 37° C. (FIG. 14, Sub-step 19), a chip in thechip rack 207 is attached to the chip head 204, and the chip head movesto the normal temperature drug solution rack 209 and sucks 1%TritonX-100 or pure water/methanol (FIG. 14, Sub-step 20).

The chip head 204 moves to a well for Testing section 4 on the firsttemperature-regulating-function-equipped plate holder (210), adds the 1%TritonX-100 or pure water/methanol (FIG. 14, Sub-step 21), and thenmoves to the dust box 214 to discard the chip.

A chip in the chip rack 207 is attached to the chip head 204, moves tothe well filled with the aforementioned reagent, sucks the whole amountof the cell suspension, and dispenses the cell suspension into thecollection plate for collection on the first plate holder 212(collection) (FIG. 14, Sub-step 22).

In the operation of the device described above, operations in the stepsof Testing sections 1 to 3 in Examples 1 and 2 are sequentiallyperformed, but the steps may be performed sequentially or in parallel.In addition, Sub-steps 1 to 18 in FIG. 14 are the same as those in FIG.13A.

[For 3D Tissue]

After the first temperature-regulating-function-equipped plate holder210 and the second temperature-regulating-function-equipped plate holder211 change from 4° C. to 37° C. (FIG. 15A, Sub-step 19), a chip in thechip rack 207 is attached to the chip head 204, and the chip head movesto the normal temperature drug solution rack 209, and sucks trypsin/EDTA(FIG. 15B, Sub-step 20).

The chip head moves to a well for Testing section 4 on the firsttemperature-regulating-function-equipped plate holder (210), adds thetrypsin/EDTA, and then the device waits at 37° C. for 30 minutes (FIG.15B, Sub-step 21). The chip head sucks 1% TritonX-100 or purewater/methanol (FIG. 15B, Sub-step 22).

The chip head 204 moves to the well for Testing section 4 on the firsttemperature-regulating-function-equipped plate holder (210), adds the 1%TritonX-100 or pure water/methanol (FIG. 15B, Sub-step 23), and thenmoves to the dust box 214 to discard the chip.

A chip in the chip rack 207 is attached to the chip head 204, moves tothe well filled with the aforementioned reagent, sucks the whole amountof the cell suspension, and dispenses the cell suspension into thecollection plate for collection on the first plate holder 212(collection) (FIG. 15B, Sub-step 24).

In the operation of the device described above, operations in the stepsof Testing sections 1 to 3 in Examples 1 and 2 are sequentiallyperformed, but the steps may be performed sequentially or in parallel.In addition, Sub-steps 1 to 18 in FIG. 15A are the same as those in FIG.13A.

<Sample Preparation: Corresponding to Steps 5 and 6 in Examples 1 and 2>

[For 2D Tissue]

After the first temperature-regulating-function-equipped plate holder210 changes from 4° C. to 37° C. (FIG. 13A, Sub-step 24), a chip in thechip rack 207 is attached to the chip head 204, the chip head moves tothe normal temperature drug solution rack (209), and sucks a buffercontaining EGTA (FIG. 13A, Sub-step 25).

The chip head 204 moves to a well for Testing section 1 on the firstplate holder 212, adds the buffer containing EGTA (FIG. 13B, Sub-step26), and then moves to the dust box 214 to discard the chip. The devicewaits at 37° C. for 30 minutes (FIG. 13B, Sub-step 26).

A chip in the chip rack 207 is attached to the chip head 204, and thechip head moves to the normal temperature drug solution rack 209, andsucks a buffer (FIG. 13B, Sub-step 27).

The chip head 204 moves to a well for Testing section 2 on the firstplate holder 212, adds the buffer (FIG. 13B, Sub-step 28), and thenmoves to the dust box 214 to discard the chip. The device waits at 37°C. for 30 minutes (FIG. 13B, Sub-step 28). A chip in the chip rack 207is attached to the chip head 204, and the chip head moves to thecold-stored drug solution rack 209, and sucks a buffer (FIG. 13B,Sub-step 29).

The chip head 204 moves to a well for Testing section 3 on the secondplate holder 213, adds the buffer (FIG. 13B, Sub-step 30), and thenmoves to the dust box 214 to discard the chip. The device waits at 4° C.for 30 minutes (FIG. 13B, Sub-step 30). A chip in the chip rack 207 isattached to the chip head 204, moves to the well filled with the EGTAbuffer containing the drug, sucks the drug-containing EGTA buffer(supernatant), and dispenses (collects) the supernatant into thecollection plate for collection on the first plate holder 212 (FIG. 13B,Sub-step 31).

A chip in the chip rack 207 is attached to the chip head 204, moves tothe well filled with the buffer containing the drug, sucks thedrug-containing buffer (supernatant), and dispenses (collects) thesupernatant into the collection plate for collection on the first plateholder 212 (FIG. 13B, Sub-step 32).

A chip in the chip rack 207 is attached to the chip head 204, moves tothe well filled with the EGTA buffer containing the drug, sucks thedrug-containing buffer (supernatant), dispenses (collects) thesupernatant into the collection plate for collection on the first plateholder 213 (FIG. 13B, Sub-step 33).

After both the first temperature-regulating-function-equipped plateholder 210 and the second temperature-regulating-function-equipped plateholder 211 change to a room temperature (FIG. 13B, Sub-step 34), a chipin the chip rack 207 is attached to the chip head 204, the chip headmoves to the normal temperature drug solution rack 209, and sucks 1%TritonX-100 or pure water/methanol (FIG. 13B, Sub-step 35).

The chip head 204 moves to the well for each of Testing sections 1, 2,and 3 on the first temperature-regulating-function-equipped plate holder(210) and the second temperature-regulating-function-equipped plateholder 211, adds the 1% TritonX-100 or pure water/methanol (FIG. 13B,Sub-step 36), and then moves to the dust box 214 to discard the chip.

A chip in the chip rack 207 is attached to the chip head 204, moves tothe well filled with the aforementioned reagent, sucks the whole amountof the cell suspension, and dispenses the cell suspension into thecollection plate for collection on the first plate holder 212 and thesecond plate holder 213 (collection) (FIG. 13B, Sub-step 37). In theoperation of the device described above, operations in the steps ofTesting sections 1 to 3 in Examples 1 and 2 are sequentially performed,but the steps may be performed sequentially or in parallel.

[For 3D Tissue]

After the first temperature-regulating-function-equipped plate holder210 changes from 4° C. to 37° C. (FIG. 16A, Sub-step 24), a chip in thechip rack 207 is attached to the chip head 204, and the chip head movesto the normal temperature drug solution rack (209), and sucks a buffercontaining trypsin/EDTA (FIG. 16B, Sub-step 25).

The chip head 204 moves to a well for Testing section 1 on the firstplate holder 212, adds the buffer containing trypsin/EDTA (FIG. 16B,Sub-step 26), and moves to the dust box 214 to discard the chip. Thedevice waits at 37° C. for 30 minutes (FIG. 16B, Sub-step 26). A chip inthe chip rack 207 is attached to the chip head 204, and the chip headmoves to the normal temperature drug solution rack 209, and sucks abuffer (FIG. 16B, Sub-step 27).

The chip head 204 moves to a well for Testing section 2 on the firstplate holder 212, adds the buffer (FIG. 16B, Sub-step 28), and thenmoves to the dust box 214 to discard the chip. The device waits at 37°C. for 30 minutes (FIG. 16B, Sub-step 28). A chip in the chip rack 207is attached to the chip head 204, and the chip head moves to thecold-stored drug solution rack 209, and sucks a buffer (FIG. 16B,Sub-step 29).

The chip head 204 moves to a well for Testing section 3 on the secondplate holder 213, adds the buffer (FIG. 16B, Sub-step 30), and thenmoves to the dust box 214 to discard the chip. The device waits at 4° C.for 30 minutes (FIG. 16B, Sub-step 30). A chip in the chip rack 207 isattached to the chip head 204, moves to the well filled with thetrypsin/EDTA buffer containing the drug, sucks the cell suspension, anddispenses (collects) the cell suspension into the collection plate forcollection on the first plate holder 212 (FIG. 16B, Sub-step 31).

The dispensed plate is subjected to centrifugation in a centrifugationunit to separate into a supernatant and a cell pellet, and then thesupernatant is dispensed (collected) into the collection plate forcollection on the first plate holder 212 (FIG. 16B, Sub-step 32).Although centrifugation is used here, the method is obviously notlimited thereto as long as it can separate the supernatant and thepellet.

A chip in the chip rack 207 is attached to the chip head 204, and thechip head 204 moves to the well filled with the buffer containing thedrug, sucks the drug-containing buffer (supernatant), and dispenses(collects) the supernatant into the collection plate for collection onthe first plate holder 212 (FIG. 16B, Sub-step 33). A chip in the chiprack 207 is attached to the chip head 204, and the chip head 204 movesto the well filled with the buffer containing the drug, sucks thedrug-containing buffer (supernatant), dispenses (collects) thesupernatant into the collection plate for collection on the first plateholder 213 (FIG. 16B, Sub-step 34).

After both the first temperature-regulating-function-equipped plateholder 210 and the second temperature-regulating-function-equipped plateholder 211 change to a room temperature (FIG. 16B, Sub-step 35), a chipin the chip rack 207 is attached to the chip head 204, and the chip head204 moves to the normal temperature drug solution rack 209, and sucks 1%TritonX-100 or pure water/methanol (FIG. 16B, Sub-step 36).

The chip head 204 moves to the well for each of Testing sections 1, 2,and 3 on the first temperature-regulating-function-equipped plate holder(210) and the second temperature-regulating-function-equipped plateholder 211, adds the 1% TritonX-100 or pure water/methanol (FIG. 16B,Sub-step 37), and then moves to the dust box 214 to discard the chip. Achip in the chip rack 207 is attached to the chip head 204, moves to thewell filled with the aforementioned reagent, sucks the whole amount of acell suspension, and dispenses the cell suspension into the collectionplate for collection on the first plate holder 212 and the second plateholder 213 (collection) (FIG. 16B, Sub-step 38).

In the operation of the device described above, operations in the stepsof Testing sections 1 to 3 in Examples 1 and 2 are sequentiallyperformed, but the steps may be performed sequentially or in parallel.In addition, Sub-steps 1 to 24 in FIG. 16A are the same as those in FIG.13A.

<Transfer from Sample Preparation Unit to Measurement Unit>

The drug collected on the culture plates is transferred to a measurementunit (FIG. 13B, Sub-step 38). In the measurement unit, the drug ismeasured by a plate reader or an LCMS (FIG. 13B, Sub-step 39).

<Calculation of Distribution Ratio and Score by Analyzing Unit andDisplay of Results>

Based on the measurement results, a distribution ratio into fractionsand a score are calculated (FIG. 13B, Sub-step 40). Then, the obtainedcalculation value is shown on the display unit (FIG. 13B, Sub-step 41).

Merely as an example of the configurations explained in Examples 1 to 3above, the following examples will be mentioned.

<Configuration 1>

It is a component analysis device, comprising a retention unit forretaining a plurality of containers for retaining a liver cell tissue,and an analysis unit for measuring a component supplied to the pluralityof containers and analyzing the component thus measured, wherein theplurality of containers are at least a first container and a secondcontainer, wherein the first container retains a first solutioncontaining a substance that releases adhesion between multiple cellsforming the liver cell tissue, and the second container retains a buffersolution, and wherein the analysis unit measures an amount of thecomponent discharged from the liver cell tissue in the first containerinto the first solution and an amount of the component discharged fromthe liver cell tissue in the second container into the buffer liquid inthe second container, and analyzes an amount of the component to bedischarged via a bile duct in the liver cell tissue.

<Configuration 2>

It is the component analysis device according to Configuration 1,further comprising a temperature regulation unit for regulating atemperature of a liquid in the plurality of containers, wherein theplurality of containers are at least a first container, a secondcontainer, and a third container, wherein the third container retainsthe buffer solution, and wherein the temperature regulation unit makesregulation so that a second temperature inside the third container islower than a first temperature inside the first container and inside thesecond container, and wherein the analysis unit measures an amount ofthe component discharged from the liver cell tissue in the thirdcontainer to the buffer solution in the third container, and analyzes anamount of the component to be discharged via a transporter of the livercell tissue and an amount of the component to be discharged from otherpaths than the transporter and the bile duct in the liver cell tissue.

<Configuration 3>

It is drug component analysis device, comprising a retention unit forretaining a plurality of containers for retaining a liver cell tissuehaving a drug absorbed therein, a liquid feeding unit for feeding aliquid in the plurality of containers, a temperature regulation unit forregulating a temperature of a liquid in the plurality of containers, andan analysis unit for measuring amounts of the drug in the plurality ofcontainers and analyzing the drug thus measured, wherein the pluralityof containers are a first container, a second container, a thirdcontainer, and a fourth container, wherein the liquid feeding unit feedsa first solution containing a substance that releases adhesion betweenmultiple cells forming the liver cell tissue into the fourth container,feeds the first solution into the first container, and feeds a buffersolution into the second container and the third container, wherein thetemperature regulation unit regulates a temperature so that atemperature inside the third container is lower than a temperatureinside the first container and inside the second container, and whereinthe analysis unit analyzes an amount of the drug discharged from theliver cell tissue in the first container to the first solution, anamount of the drug discharged from the liver cell tissue in the secondcontainer to the buffer solution in the second container, an amount ofthe drug discharged from the liver cell tissue in the third container tothe buffer solution in the third container, an amount of the drugdischarged from the liver cell tissue in the fourth container to thebuffer solution in the fourth container, an amount of the drug to bedischarged from a transporter in the liver cell tissue, an amount of thedrug to be discharged from a bile duct in the liver cell tissue, and anamount of the component to be discharged from other paths than thetransporter and the bile duct in the liver cell tissue.

<Configuration 4>

It is a component analysis method, including an analyzing step formeasuring a component fed into a plurality of containers retaining aliver cell tissue and analyzing the component thus measured, wherein theplurality of containers are at least a first container and a secondcontainer, wherein the first container retains a first solutioncontaining a substance that releases adhesion between multiple of cellsforming the liver cell tissue, and wherein the second container retainsa buffer solution, and wherein in the analyzing step, an amount of thecomponent discharged from the liver cell tissue in the first containerinto the first solution and an amount of the component discharged fromthe liver cell tissue in the second container into the buffer solutionin the second container are measured and an amount of the component tobe discharged via a bile duct in the liver cell tissue is analyzed.

<Configuration 5>

It is the component analysis method according to the configuration 4,further comprising a temperature regulating step for regulating atemperature of a liquid in the plurality of containers, wherein theplurality of containers are at least the first container, the secondcontainer, and a third container, wherein the third container retainsthe buffer solution, and wherein in the temperature regulating step, asecond temperature inside the third container is regulated to be lowerthan a first temperature inside the first container and inside thesecond container, and wherein in the analyzing step, an amount of thecomponent discharged from the liver cell tissue in the third containerinto the buffer solution in the third container is measured, and anamount of the component to be discharged via a transporter in the livercell tissue and an amount of the component to be discharged from otherpaths than the transporter and the bile duct in the liver cell tissueare analyzed.

<Configuration 6>

It is a drug component analysis method, comprising a liquid feeding stepfor feeding a liquid in a plurality of containers retaining a liver celltissue having a drug absorbed therein, a temperature regulating step forregulating a temperature of a liquid in the plurality of container, andan analyzing step for measuring amounts of the drug in the plurality ofcontainers and analyzing the drug thus measured, wherein the pluralityof containers are a first container, a second container, a thirdcontainer, and a fourth container, wherein in the liquid feeding step, afirst solution containing a substance that releases adhesion betweenmultiple cells forming the liver cell tissue is fed to the fourthcontainer, the first solution is fed to the first container, and abuffer solution is fed to the second container and the third container,wherein in the temperature regulating step, a temperature is regulatedso that the temperature inside the third container becomes lower than atemperature inside the first container and inside the second container,and wherein in the analyzing step, an amount of the drug discharged fromthe liver cell tissue in the first container into the first solution, anamount of the drug discharged from the liver cell tissue in the secondcontainer into the buffer solution in the second container, an amount ofthe drug discharged from the liver cell tissue in the third containerinto the buffer solution in the third container, an amount of the drugdischarged from the liver cell tissue in the fourth container into thebuffer solution in the fourth container, an amount of the drug to bedischarged from a transporter in the liver cell tissue, an amount of thedrug to be discharged from a bile duct in the liver cell tissue, and anamount of the component to be discharged from other paths than thetransporter and the bile duct in the liver cell tissue are analyzed.

REFERENCE SIGNS LIST

-   001 culture plate-   002 well-   106 culturing unit-   107 sample preparation unit-   107A input unit-   107B temperature regulation unit-   107C liquid feeding unit-   107D centrifugation unit-   108 analysis unit-   108A measurement unit-   108B analyzing unit-   109 display unit-   201 syringe pump controller-   202 temperature regulation controller-   203 syringe pump-   204 chip head-   205 suction head-   206 waste liquid tank-   207 chip rack-   208 cold-stored drug solution rack-   209 normal temperature drug solution rack-   210 first temperature-regulating-function-equipped plate holder-   211 second temperature-regulating-function-equipped plate holder-   212 first plate holder-   213 second plate holder-   214 dust box-   215 circulator-   216 suction pump-   301 sucked liquid-   302 chip

The invention claimed is:
 1. A component analysis method, comprising:providing a plurality of containers including a first container and asecond container, wherein the first container comprises contents ofliver cell tissue comprising a 3D culture and a first solutioncontaining a first substance that releases adhesion between multiplecells that form the liver cell tissue and that allows a bile duct formedin the liver cell tissue to collapse, and a substance that comprisestrypsin and EDTA for cutting the 3D culture liver cell tissue intosingle sells, wherein a temperature in the first container is maintainedat 37° C., such that a fraction discharged from cells into a bloodvessel side includes a fraction via passive diffusion and a fraction viaa transporter, and the second container comprises contents of liver celltissue comprising a 3D culture and a buffer solution; contacting thecontents of the first container and the contents of the second containerwith a component; measuring an amount of the component discharged fromthe liver cell tissue in the first container into the first solution;measuring an amount of the component discharged from the liver celltissue in the second container into the buffer solution in the secondcontainer; and measuring an amount of the component to be discharged viaa bile duct in the liver cell tissue.
 2. The component analysis methodaccording to claim 1, further comprising: regulating a temperature of aliquid in a third container and in the first container and the secondcontainer, such that a second temperature inside the third container islower than a first temperature inside the first container and inside thesecond container, wherein the third container contains the buffersolution; and measuring an amount of the component discharged from theliver cell tissue in the third container into the buffer solution in thethird container; measuring an amount of the component to be dischargedvia a transporter in the liver cell tissue; and measuring an amount ofthe component to be discharged from paths other than the transporter andthe bile duct in the liver cell tissue.
 3. A drug component analysismethod, comprising feeding a liquid into a plurality of containers eachcontaining liver cell tissue comprising a 3D culture having a drugabsorbed therein, regulating a temperature of respective liquids in theplurality of containers, and measuring an amount of the drug in theplurality of containers and analyzing the drug thus measured, whereinthe plurality of containers include a first container, a secondcontainer, a third container, and a fourth container, wherein in theliquid feeding step, a first solution containing a substance thatreleases adhesion between multiple cells forming the liver cell tissueand that allows a bile duct formed in the liver cell tissue to collapse,and a substance that comprises trypsin and EDTA for cutting the 3Dculture liver cell tissue into single sells is fed in the firstcontainer, wherein a temperature in the first container is maintained at37° C., such that a fraction discharged from cells into a blood vesselside includes a fraction via passive diffusion and a fraction via atransporter, and a buffer solution is fed to the second container andthe third container, wherein in the temperature regulating step, thetemperatures of the plurality of containers are regulated so that atemperature inside the third container is lower than a temperatureinside the first container and inside the second container, and whereinthe method further comprises: measuring an amount of the drug dischargedfrom the liver cell tissue in the first container into the firstsolution, measuring an amount of the drug discharged from the liver celltissue in the second container into the buffer solution in the secondcontainer, measuring an amount of the drug discharged from the livercell tissue in the third container into the buffer solution in the thirdcontainer, measuring an amount of the drug discharged from the livercell tissue in the fourth container into the buffer solution in thefourth container, measuring an amount of the drug to be discharged froma transporter in the liver cell tissue, measuring an amount of the drugto be discharged from a bile duct in the liver cell tissue, andmeasuring an amount of the drug to be discharged from paths other thanthe transporter and the bile duct in the liver cell tissue.
 4. Thecomponent analysis method according to claim 1, further comprising thestep of determining a distribution ratio into fractions of the componentbased on at least one of the measured amount of the component dischargedfrom the liver cell tissue in the first container into the firstsolution and the measured amount of the component discharged from theliver cell tissue in the second container into the buffer solution inthe second container.
 5. The drug component analysis method according toclaim 3, further comprising the step of determining a distribution ratiointo fractions of the component based on at least one of the measuredamount of the drug discharged from the liver cell tissue in the firstcontainer into the first solution and the measured amount of the drugdischarged from the liver cell tissue in the second container into thebuffer solution in the second container.